Knockout serum

Cells were cultured in DMEM-F12 (Invitrogen-Life Technologies), together with basic fibroblast growth factor (bFGF) (10 ng/mL), epidermal growth factor (EGF) (20 ng/mL), leukemia inhibitory factor (LIF) (10 ng/mL) and 10% knockout serum (all from Sigma). Two-hundred μL of the medium containing 2000 cells/well were plated in 96-well low attachment culture plates. Those spheres with a diameter of ≥100 μm within each well were counted after 10 days of culture. Images were captured using a Carl Zeiss microscope.

46C ESCs (expressing Sox1-GFP) derived from strain 129Ola mice were cultured on 0.1% gelatin-coated dishes in Glasgow minimal essential medium (Sigma), 10% knockout serum replacement, 1% modified Eagle’s medium (MEM) non-essential amino acids, 1 mM sodium pyruvate (Life Technologies), 0.1 mM 2-mercaptoethanol (Sigma), and 100 U/ml recombinant human leukemia inhibitory factor). hiPS4 and SA181 human ESC lines purchased from Cellartis and maintained in DEF-CS (Cellartis). Human neural differentiation was performed as described previously (Chambers et al., 2009 (link)), except that cells were seeded at a density of 6 × 10⁴ cells/cm² on Matrigel (20 μg/cm²) and grown for 48 hrs in cell medium before switching to differentiation media. Murine neural differentiation was performed as described previously (Stavridis et al., 2007 (link), Ying et al., 2003 (link)). 0.5–1.5 × 10⁴/cm² ESCs were plated on 0.1% gelatin-coated dishes in N2B27 (DMEM/F12; Gibco) supplemented with modified N2 (25 μg/ml insulin, 100 μg/ml apo-transferrin, 6 ng/ml progesterone, 16 μg/ml putrescine, 30 nM sodium selenite and 50 μg/ml BSA fraction V; Gibco). Medium was renewed every 2 days.